Characterizing and Quantitating Therapeutic Tethered Multimeric Antibody Degradation Using Affinity Capture Mass...

Area of Science:

• Biopharmaceutical analysis
• Protein therapeutics characterization
• Mass spectrometry applications

Background:

• Protein therapeutics face pharmacokinetic challenges, including biotransformation and degradation at fusion/conjugation sites in multivalent formats.
• Degradation of tethered antibody formats results in large mass differences, causing mass spectrometry response bias and inaccurate stability quantitation.
• Existing methods struggle to accurately quantify degradation products due to inherent biases in mass spectrometry response.

Purpose of the Study:

• To develop and validate an assay strategy for accurate characterization and quantitation of degradations in multivalent antibody formats.
• To address and correct for mass spectrometry response bias observed in degraded multivalent antibody species.
• To enable reliable stability profiling of engineered antibody formats in biological matrices.

Main Methods:

• Incorporated response bias corrections into an assay strategy for multivalent antibodies.
• Spiked selected tethered multivalent antibodies and IgG antibodies in serum at known ratios.
• Generated and qualified calibration curves using a five-parameter logistic fit for MS ion response ratios against known spiked-in ratios.

Main Results:

• Observed significant response differences (30-80%) between intact and cleaved multivalent antibody products.
• Achieved accurate calibration standards with coefficients of variation (CVs) < 8%.
• Validated the assay for measuring degradations with accuracy within 8% (5% degradation) and 2% (15% degradation).

Conclusions:

• The developed assay strategy effectively corrects for mass spectrometry response bias in multivalent antibody degradation analysis.
• This method enables accurate quantitation of stability profiles for multivalent tethered antibody formats.
• The approach is applicable to both in vitro serum stability and pharmacokinetic study samples, advancing biotherapeutic characterization.